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Objectives To investigate the in vitro activity of fosfomycin, colistin and combinations thereof against planktonic and biofilm cultures of Gram-negative pathogens, mostly showing MDR phenotypes, at concentrations achievable via inhalation of aerosolized drugs. Methods Activity against planktonic cultures was tested by the chequerboard assay with 130 strains, including 52 Pseudomonas aeruginosa, 47 Klebsiella pneumoniae, 19 Escherichia coli, 7 Stenotrophomonas maltophilia and 5 Acinetobacter baumannii. Activity against biofilm cultures was tested by biofilm chequerboard and quantitative antibiofilm assays with a subset of 20 strains. In addition, 10 of these strains were tested in mutant prevention concentration (MPC) assays. Results Against planktonic cultures, synergism between fosfomycin and colistin was detected with a minority (10%) of strains (eight K. pneumoniae and five P. aeruginosa), while antagonism was never observed. Synergism between fosfomycin and colistin against biofilms was observed with the majority of tested strains (16/20 in biofilm chequerboard assays, and 18/20 in the quantitative antibiofilm assays), including representatives of each species and regardless of their resistance genotype or phenotype. Furthermore, combination of fosfomycin and colistin was found to significantly reduce the MPC of individual drugs. Conclusions Fosfomycin and colistin in combination, at concentrations achievable via inhalation of nebulized drugs, showed notable synergy against MDR Gram-negative pathogens grown in biofilm, and were able to reduce the emergence of fosfomycin- and colistin-resistant subpopulations.

The antiarrhythmic sodium-channel blocker mexiletine is used to treat patients with myotonia. However, around 30% of patients do not benefit from mexiletine due to poor tolerability or suboptimal response. Safinamide is an add-on therapy to levodopa for Parkinson's disease. In addition to MAOB inhibition, safinamide inhibits neuronal sodium channels, conferring anticonvulsant activity in models of epilepsy. Here, we investigated the effects of safinamide on skeletal muscle hNav1.4 sodium channels and in models of myotonia, in-vitro and in-vivo. Using patch-clamp, we showed that safinamide reversibly inhibited sodium currents in HEK293T cells transfected with hNav1.4. At the holding potential (hp) of −120 mV, the half-maximum inhibitory concentrations (IC50) were 160 and 33 μM at stimulation frequencies of 0.1 and 10 Hz, respectively. The calculated affinity constants of safinamide were dependent on channel state: 420 μM for closed channels and 9 μM for fast-inactivated channels. The p.F1586C mutation in hNav1.4 greatly impaired safinamide inhibition, suggesting that the drug binds to the local anesthetic receptor site in the channel pore. In a condition mimicking myotonia, i.e. hp. of −90 mV and 50-Hz stimulation, safinamide inhibited INa with an IC50 of 6 μM, being two-fold more potent than mexiletine. Using the two-intracellular microelectrodes current-clamp method, action potential firing was recorded in vitro in rat skeletal muscle fibers in presence of the chloride channel blocker, 9-anthracene carboxylic acid (9-AC), to increase excitability. Safinamide counteracted muscle fiber hyperexcitability with an IC50 of 13 μM. In vivo, oral safinamide was tested in the rat model of myotonia. In this model, intraperitoneal injection of 9-AC greatly increased the time of righting reflex (TRR) due to development of muscle stiffness. Safinamide counteracted 9-AC induced TRR increase with an ED50 of 1.2 mg/kg, which is 7 times lower than that previously determined for mexiletine. In conclusion, safinamide is a potent voltage and frequency dependent blocker of skeletal muscle sodium channels. Accordingly, the drug was able to counteract abnormal muscle hyperexcitability induced by 9-AC, both in vitro and in vivo. Thus, this study suggests that safinamide may have potential in treating myotonia and warrants further preclinical and human studies to fully evaluate this possibility.

To investigate whether the reversible MAO-B inhibitor and sodium channel blocker safinamide impairs glutamate release under parkinsonian conditions in vivo, and this effect is dependent on MAO-B inhibition, safinamide (and rasagiline as a comparator) were administered to 6-hydroxydopamine hemilesioned rats, a model of Parkinson's disease, and haloperidol-treated rats, a model of neuroleptic-induced parkinsonism. A microdialysis probe was implanted in the dopamine-depleted dorsolateral striatum, globus pallidus, subthalamic nucleus or substantia nigra reticulata of 6-hydroxydopamine hemilesioned rats. Glutamate and GABA release was stimulated by reverse dialysis of veratridine, and safinamide or rasagiline were acutely administered before veratridine at doses inhibiting MAO-B > 50%. A microdialysis probe was implanted in the substantia nigra reticulata of naive rats to monitor glutamate and GABA release following acute haloperidol and safinamide administration. Safinamide inhibited the veratridine-evoked glutamate release in the globus pallidus and subthalamic nucleus but not in the striatum and substantia nigra. Moreover, it reduced pallidal and nigral GABA release. Conversely, rasagiline failed to modify the veratridine-induced glutamate and GABA release in the basal ganglia. Safinamide also inhibited the haloperidol-induced nigral glutamate release. MAO-B inhibitors safinamide and rasagiline differ in their abilities to inhibit depolarization-evoked glutamate release in the basal ganglia of parkinsonian rats. The ineffectiveness of rasagiline suggests that MAO-B inhibition does not contribute to the anti-glutamatergic activity of safinamide. The glutamate-inhibiting action of safinamide within the subthalamo-external pallidal loop, which shows abnormal activity in Parkinson's disease, might contribute to its therapeutic actions of improving motor performance without provoking troublesome dyskinesia.

Safinamide (Xadago) is a novel dual-mechanism drug that has been approved in the European Union and United States as add-on treatment to levodopa in Parkinson's disease therapy. In addition to its selective and reversible monoamine oxidase B inhibition, safinamide through use-dependent sodium channel blockade reduces overactive glutamatergic transmission in basal ganglia, which is believed to contribute to motor symptoms and complications including levodopa-induced dyskinesia (LID). The present study investigated the effects of safinamide on the development of LID in 6-hydroxydopamine (6-OHDA)-lesioned rats, evaluating behavioral, molecular, and neurochemical parameters associated with LID appearance. 6-OHDA-lesioned rats were treated with saline, levodopa (6 mg/kg), or levodopa plus safinamide (15 mg/kg) for 21 days. Abnormal involuntary movements, motor performance, molecular composition of the striatal glutamatergic synapse, glutamate, and GABA release were analyzed. In the striatum, safinamide prevented the rearrangement of the subunit composition of N-methyl-D-aspartate receptors and the levodopa-induced increase of glutamate release associated with dyskinesia without affecting the levodopa-stimulated motor performance and dyskinesia. Overall, these findings suggest that the striatal glutamate-modulating component of safinannide's activity may contribute to its clinical effects, where its long-term use as levodopa add-on therapy significantly improves motor function and "on" time without troublesome dyskinesia.

Neuronal nicotinic receptors (nAChRs) are a heterogeneous family of ion channels differently expressed in the nervous system where, by responding to the endogenous neurotransmitter acetylcholine, they contribute to a wide range of brain activities and influence a number of physiological functions. Over recent years, the application of newly developed molecular and cellular biological techniques has made it possible to correlate the subunit composition of nAChRs with specific nicotine-elicited behaviours, and refine some of the in vivo physiological functions of nAChR subtypes. The major new findings are the widespread expression of nAChRs, outside the nervous system, their specific and complex organisation, and their relevance to normal brain function. Moreover, the combination of clinical and basic research has better defined the involvement of nAChRs in a growing number of nervous pathologies other than degenerative diseases. However, there are still only a limited number of nicotinic-specific drugs and, although some nicotinic agonists have an interesting pharmacology, their clinical use is limited by undesirable side effects. Some selective nicotinic ligands have recently been developed and used to explore the complexity of nAChR subtype structure and function in the expectation that they will become rational therapeutic alternatives in a number of neurodegenerative, neuropsychiatric and neurological disorders. In this review, we will discuss the molecular basis of brain nAChR structural and functional diversity mainly in pharmacological and biochemical terms, and summarise current knowledge concerning the newly discovered drugs used to classify the numerous receptor subtypes and treat the brain diseases in which nAChRs are involved.

Chronic and acute exposure to organophosphate (OP) pesticides may lead to persistent neurological and neurobehavioral effects, which cannot be explained by acetylcholinesterase (AChE) inhibition alone. It is suggested that other brain proteins are involved. Effects of commonly used organophosphate pesticides on rat neuronal α4β2 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes have been investigated using the two-electrode voltage clamp technique. Several OP pesticides, e.g., parathion-ethyl, chlorpyrifos and disulfoton, inhibited the ACh-induced ion current with potencies in the micromolar range. The potency of inhibition increased with increasing concentrations of the agonist ACh. Comparison of the potency of nAChR inhibition with the potency of AChE inhibition demonstrated that some OPs inhibit nAChRs more potently than AChE. Binding experiments on α 4β2 nAChRs showed that the OPs noncompetitively interact with nAChRs. The inhibitory effects on nAChRs are adequately described and explained by a sequential two-step mechanism, in which rapidly reversible OP binding to a separate binding site leads to inhibition followed by a stabilization of the blocked state or receptor desensitization. It is concluded that OPs interact directly with neuronal α4β2 nAChRs to inhibit the agonist-induced response. This implicates that neuronal α4β2 nAChRs are additional targets for some OP pesticides. © Society of Toxicology 2004; all rights reserved. Chemicals / CAS: chlorpyrifos, 2921-88-2; dipeptidyl carboxypeptidase, 9015-82-1; disulfoton, 298-04-4; parathion, 3270-86-8, 56-38-2, 597-88-6; Bicyclo Compounds, Heterocyclic; Cholinesterase Inhibitors; epibatidine, 140111-52-0; Insecticides; Ligands; Nicotinic Agonists; Nicotinic Antagonists; Organophosphorus Compounds; Pyridines; Receptors, Nicotinic

Introduction Vaginal dryness due to vaginal atrophy is a common complaint of postmenopausal women, interfering with sexual function and quality of life. Hormone replacement therapy is the only effective therapy but with known risks that leave unmet medical needs. A new product, ZP-025 vaginal gel, containing purified (dialyzed lyophilized) bovine colostrum, has been developed for the treatment of vaginal dryness secondary to vaginal atrophy. Aim The study aims to investigate the effects of intravaginal application of ZP-025 on vaginal atrophy using an animal model. Methods Ovariectomized female Sprague-Dawley rats were used. Three weeks after surgery, rats were divided into four groups and treated for 4 weeks (twice a day) with placebo or ZP-025 at low (0.5%) or high (2.3%) concentrations of colostrum; in the control group, rats did not receive any treatment. Changes in vaginal blood flow due to pelvic nerve stimulation were assessed by laser Doppler flowmetry and vaginal tissue was collected for histological assay. Main Outcome Measures The main outcome measures were vaginal blood flow before and after pelvic nerve stimulation and histology of vaginal epithelium. Results Treatment with ZP-025 to ovariectomized rats induced an increase of vaginal blood flow parameters (vascular capacitance, amplitude and area under the curve of the response) in response to pelvic nerve stimulation compared with control group, statistically significant at 2.3%. Vaginal epithelium showed a physiological estrous cycle aspect in treated animals, with at least five cell layers vs. one or two cell layers in control rats. As expected from a topical formulation, systemic effects on body weights and uterine wet weights were not observed with application of ZP-025. Conclusions In this study, the new product ZP-025, containing purified colostrum, was shown to have beneficial effects on vaginal atrophy in ovariectomized rats, improving vaginal hemodynamics and thickness of vaginal epithelium.

Retina from 1-day-old chicks is a valuable tissue model for studying neuronal nicotinic receptors because it expresses a large number of the developmentally regulated high affinity [ 3 H]epibatidine labeled nicotinic receptors. Most of these receptors contain the β4 subunit associated with different α subunits. Using a sequential immunodepletion procedure with anti-α6, anti-β3, anti-β2, and anti-β4 antibodies, we purified an α4β4 nicotinic receptor subtype that accounts for approximately 20 to 25% of the high affinity [ 3 H]epibatidine labeled receptors present in retina at that developmental time. Immunoprecipitation and Western blotting experiments confirmed that the purified subtype contains only the α4 and β4 subunits. This receptor binds a number of agonists and the antagonist dihydro-β-erythroidine with nanomolar affinity, whereas it has micromolar affinity for the α-conotoxin MII and methyllycaconitine toxins and other nicotinic antagonists. Comparison of the pharmacological profile of this purified native subtype with that of the same subtype transiently expressed in human BOSC23 cells showed that they have very similar rank orders and absolute K i values for several nicotinic drugs. Finally, because chick retina expresses an α6β4-containing subtype with a high affinity for the α-conotoxin MII, we used native and transfected α4β4 and α6β4 subtypes to investigate the relative contributions of the α and β subunits to this binding, and found that the α6 subunit determines the high affinity for this toxin.

The most widely expressed neuronal nicotinic acetylcholine receptor subtype in chick brain is that containing the α4 and β2 subunits. However, immunoprecipitation and localization studies have shown that some brain areas also contain the α2 and/or α5 subunits, whose role in the definition of receptor properties is still intriguing. Using subunit-specific polyclonal antibodies, we found that the optic lobe is the chick central nervous system region that expresses the highest level of α2-containing receptors. Immunoprecipitation studies of these immunopurified α2-containing receptors labeled with the nicotinic agonist [ 3 H]epibatidine showed that almost all of them contained the β2 subunit and that more than 66% contained the α5 subunit. Western blot analyses of the purified receptors confirmed the presence of the α2, α5, and β2 subunits and the absence of the α3, α4, α6, α7, α8, β3, and β4 subunits. The α2-containing receptors are developmentally regulated: their expression increases 25 times from embryonic day 7 to posthatching day 1 in the optic lobe, compared with an increase of only 5-fold in the forebrain. The α2-containing optic lobe receptors bind [ 3 H]epibatidine ( K d = 29 pM) and a number of other nicotinic agonists with very high affinity and have a pharmacological profile very similar to that of the α4β2 subtype. They form functional cationic channels when reconstituted in lipid bilayers, with pharmacological and biophysical properties different from those of the α4β2 subtype. These channels are activated by nicotinic agonists in a dose-dependent manner and are blocked by the nicotinic antagonist d -tubocurarine.

s / Parkinsonism and Related Disorders 22 (2016) e149ee192 e177 Sciences, University of Ferrara, Ferrara, Italy; Consultant, Gallarate, Varese, Italy; RD Department of Medical Sciences, University of Ferrara, Ferrara, Italy; Consultant, Gallarate, Varese, Italy Objectives: Safinamide (Xadago®) is a novel drug recently approved as add-on therapy to levodopa in midto late-stage Parkinson's disease. Xadago® shows a unique dual mechanism of action: it has both dopaminergic properties (highly selective, potent and reversible inhibition of monoamine oxidase-B) and non-dopaminergic properties (state and usedependent sodium channel blockade causing inhibition of excessive glutamate release). The study aim was to evaluate the effect of safinamide on stimulated glutamate release in rat hippocampus at doses giving free brain concentrations able to block sodium channels and to provide a PK/PD approach to support its clinical relevance on overactive glutamate release inhibition. Methods: Glutamate release, stimulated by veratridine in the hippocampus of rats, was measured by in vivo microdialysis. Results: Safinamide was able to significantly inhibit the glutamate release in a dose-dependent manner and the relative measured free brain concentrations (0.3-2.2 mM) support the inhibition of sodium channels (2050% Ki. A functional blockade of sodium channels suggests that pharmacodynamically active concentrations of safinamide are available at the target sites in the brain: in rodents significant protection (50-70%) from convulsions was found when free brain concentration of 0.37-1.4 mM was reached. At the recommended clinical doses (50 and 100 mg) the estimated human free brain safinamide concentrations (0.21-1.92 mM) overlap the range of measured free brain concentrations effective in inhibiting overactive glutamate release in animals. Conclusions: These results clearly support the non-dopaminergic aspects of Xadago® mode of action in rats, likely relevant also in humans. P 6.004. 4I (N-(3-CHLORO-2-METHYLPHENYL) QUINOXALIN-2-CARBOXAMIDE), A NOVEL 5-HT3 RECEPTOR ANTAGONIST ALLEVIATES DEPRESSIVE BEHAVIOR EVOKED IN STREPTOZOTOCIN-INDUCED DIABETIC MICE: ROLE OF OXIDATIVE STRESS Deepali Gupta, Mahesh Radhakrishnan. Department of Pharmacy, Pilani

Antibodies raised against human alpha 2-6 and beta 2-4 nicotinic receptor subunits were utilized to fractionate H-3-epibatidine binding in human temporal cortex and striatum. The predominant receptor subtypes in both regions contained alpha 4 and beta 2 subunits. In normal cortex, 10% of binding was also associated with alpha 2 subunits, whereas in the striatum, contributions by alpha 6 (17%) and beta 3 (23%) were observed. Minimal binding (

Nicotinic acetylcholine receptors (nAChRs) represent an important modulator of striatal function both under normal conditions and in pathological states such as Parkinson's disease. Because different nAChR subtypes may have unique functions, immunoprecipitation and ligand binding studies were done to identify their subunit composition. As in the rodent, alpha2, alpha4, alpha6, beta2, and beta3 nAChR subunit immunoreactivity was identified in monkey striatum. However, distinct from the rodent, the present results also revealed the novel presence of alpha3 nAChR subunit-immunoreactivity in this same region, but not that for alpha5 and beta4. Relatively high levels of alpha2 and alpha3 subunits were also identified in monkey cortex, in addition to alpha4 and beta2. Experiments were next done to determine whether striatal subunit expression was changed with nigrostriatal damage. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment decreased alpha6 and beta3 subunit immunoreactivity by approximately 80% in parallel with the dopamine transporter, suggesting that they are predominantly expressed on nigrostriatal dopaminergic projections. In contrast, alpha3, alpha4, and beta2 subunit immunoreactivity was decreased approximately 50%, whereas alpha2 was not changed. These data, together with those from dual immunoprecipitation and radioligand binding studies ([(3)H]cytisine, (125)I-alpha-bungarotoxin, and (125)I-alpha-conotoxin MII) suggest the following: that alpha6beta2beta3, alpha6alpha4beta2beta3, and alpha3beta2* nAChR subtypes are present on dopaminergic terminals and that the alpha4beta2 subtype is localized on both dopaminergic and nondopaminergic neurons, whereas alpha2beta2* and alpha7 receptors are localized on nondopaminergic cells in monkey striatum. Overall, these results suggest that drugs targeting non-alpha7 nicotinic receptors may be useful in the treatment of disorders characterized by nigrostriatal dopaminergic damage, such as Parkinson's disease.

Homochiral E and Z isomers of N-methylprolinal O-isopropyloxime and (1-methyl-2-pyrrolidinyl)methoxyimines were synthesized as candidate bioisosteres of nicotine and its isoxazolic analogue ABT 418. Two of them, namely (S)-2-isopropylideneaminooxymethyl- and (Z)-(S)-2-ethylideneaminooxymethyl-1-methylpyrrolidine, proved to bind at α4β2 nicotinic acetylcholine receptor with submicromolar affinity and remarkable selectivity over α7 and muscarinic receptors thus supporting the hypothesized bioisosteric relationship between their methyloxyimino group and the aromatic heterocycles of the reference ligands.

By acting through retinal nicotinic acetylcholine receptors (nAChRs), acetylcholine plays an important role in the development of both the retina and central visual pathways. Ligand binding and immunoprecipitation studies with subunit-specific antibodies showed that the expression of alphaBungarotoxin (alphaBgtx) and high-affinity epibatidine (Epi) receptors is regulated developmentally and increases until postnatal day 21 (P21). The increase in Epi receptors is caused by a selective increase in the subtypes containing the alpha2, alpha4, alpha6, beta2, and beta3 subunits. Immunopurification studies revealed three major populations of Epi receptors on P21: alpha6(*) receptors (26%), which contain the alpha6beta3beta2, alpha6alpha4beta3beta2, and alpha6alpha3/alpha2beta3beta2 subtypes; alpha4(non-alpha6)(*) receptors (60%), which contain the alpha2alpha4beta2 and alpha4beta2 subtypes; and (non-alpha4/non-alpha6)(*) receptors (14%), which contain the alpha2beta2/beta4 and alpha3beta2/beta4 subtypes. These three populations can be pharmacologically discriminated using alphaconotoxin MII, which binds the alpha6(*) population with high affinity. In situ hybridization showed that the transcripts for all of the subunits are heterogeneously distributed throughout retinal neurons at P21, with alpha3, alpha6, and beta3 transcripts preferentially concentrated in the ganglion cell layer, alpha5 in the inner nuclear layer, and alpha4 and beta2 distributed rather homogeneously. To investigate whether nAChR expression is affected by visual experience, we also studied dark-reared P21 rats. Visual deprivation had no effect on the expression of alphaBgtx receptors or the developmentally regulated Epi receptors containing the alpha2, alpha6, and/or beta3 subunits but significantly increased the expression of the Epi receptors containing the alpha4 and beta2 subunits. Overall, this study demonstrates that the retina is the rat neural region that expresses the widest array of nAChR subtypes. These receptors have a specific distribution, and their expression is finely regulated during development and by visual experience.

Although the neuronal nicotinic beta3 subunit was cloned several years ago, it has only recently been shown to form heteromeric channels when associated with other nicotinic subunits, and very little information is available concerning its assembly in the native nicotinic receptors of the nervous system. Using subunit-specific antibodies and immunoprecipitation experiments, we have identified the retina as being the chick central nervous system (CNS) area that expresses the highest level of the beta3 subunit. Sequential immunopurification experiments showed that there are at least two populations of beta3-containing receptors in chick retina: in one, the beta3 subunit is associated with the alpha6 and beta4 subunits; in the other more heterogeneous population, the beta3 subunit is associated with the alpha2, alpha3, alpha4, beta2 and beta4 subunits. Both of these receptor populations bind [3H]epibatidine and a number of nicotinic receptor agonists with high affinity (nM) and nicotinic receptor antagonists with a lower affinity (microM). The greatest pharmacological difference between the two populations is the affinity for the alpha-conotoxin MII, which inhibits binding to alpha6-containing receptors and not that to beta3-containing receptors. We also searched for the presence of the beta3 subunit associated with the alpha-bungarotoxin binding subunits alpha7 and/or alpha8 in retina and chick brain. Immunoprecipitation studies using anti-beta3 antibodies did not detect any specific alpha-bungarotoxin labeled receptors, thus, indicating that the beta3 subunit is not present in the alpha-bungarotoxin receptors of these areas.

Despite the fact that the neuronal chick alpha6 subunit was first cloned several years ago and recently has been shown to form acetylcholine (ACh)-activated channels in heterologous systems, no information is yet available concerning the structure and function of the alpha6-containing nicotinic receptors in neuronal tissues. Using subunit-specific antibodies directed against two different epitopes of the chick alpha6 subunit, we performed immunoprecipitation experiments on immunopurified alpha6-containing receptors radiolabeled with the nicotinic agonist [3H]epibatidine (Epi): almost all of the alpha6 receptors contained the beta4 subunit, 51% the beta3 subunit, 42% the alpha3 subunit, and 7.5% the beta2 subunit. Western blot analyses of the purified receptors confirmed the presence of the alpha3, beta3, beta2, and beta4 subunits, and the absence of the alpha4, alpha5, and alpha7 subunits. The alpha6-containing receptors bind [3H]Epi (Kd = 35 pM) and a number of other nicotinic agonists with very high affinity, the rank order being Epicytisinenicotine1, 1-dimethyl-4-phenylpiperaziniumacetylcholinecarbamylcholine. The alpha6 receptors also have a distinct antagonist pharmacological profile with a rank order of potency of alpha-conotoxin MIImethyllycaconitinedihydro-beta-erythroydineMG624d-tubocurarinedecamethoniumhexamethonium. When reconstituted in lipid bilayers, the alpha6-containing receptors form functional cationic channels with a main conductance state of 48 pS. These channels are activated by nicotinic agonists in a dose-dependent manner, and blocked by the nicotinic antagonist d-tubocurarine.